The proposed studies continue to investigate the regulation of cell differentiation. The emphasis is on early cell physiological responses to inducers of terminal differentiation. The potential regulatory role of the RB (retinoblastoma) gene will be focused on. The insight so gained will hopefully promote the potential clinical use of differentiation inducing agents to treat hematopoietic neoplastic disease. Chemotherapy of leukemia has historically relied on relatively toxic agents to kill the tumor stem cells. An alternative is to use potentially less toxic agents to induce the terminal differentiation of leukemia cells, thereby arresting their proliferative activity and potentially restoring some of the differentiated cells that the patient is otherwise deprived of. To be able to optimally exploit this new treatment modality, it becomes necessary to know how various agents act at the cellular and molecular level to effect terminal differentiation of neoplastic cells. We will study this using a human leukemia cell line which differentiates in vitro. One target regulatory gene that has been little studied in this regard is the tumor suppressor genes, of which the RB retinoblastoma gene is the prototype. Expression of this gene has an apparent regulatory role in effecting terminal differentiation. Thus it becomes of interest to assessing tumor response and drug efficacy to determine how RB is regulated during induced terminal differentiation and how such agents affect its expression. Molecular mechanisms of differentiation induction therapy involving the RB tumor suppressor gene's role in differentiation will thus be studied. The working hypothesis is that the RB gene has a regulatory role in effecting terminal differentiation. Our preliminary evidence indicates that RB expression is down-regulated anteceding onset of terminal differentiation. Using a variety of agents to induce terminal differentiation of HL-60 human promyelocytic leukemia cells as an in vitro model, the role of the RB gene in effecting induced terminal differentiation will be studied by characterizing regulation of its expression and then experimentally manipulating that expression by molecular biological techniques to determine the consequences on cellular ability to differentiate. Because differentiation can be induced at will in these cells, they provide a powerful tool for investigating this process.